Composite Nanoparticles And Uses Thereof

ABSTRACT

Provided herein are composite nanoparticles, methods of making composite nanoparticles and methods of using composite nanoparticles to treat or ameliorate various diseases, such as, for example, cancer.

This application claims priority under 35 U.S.C. § 119 (e) from U.S. Provisional Application Ser. No. 62/305,526 filed Mar. 8, 2016. application Ser. No. 15/453,814 filed, Mar. 8, 2015, which are hereby incorporated by reference in their entirety.

FIELD

Provided herein are composite nanoparticles, methods of making composite nanoparticles and methods of using composite nanoparticles to treat, prevent or ameliorate various medical disorders, such as, for example, cancer.

BACKGROUND

As is well known, VEGF tumor vasculature, which is driven by tumor hypoxia, differs significantly from vasculature found in normal blood vessels. Importantly, tumor VEGF vasculature results in significant gaps between individual tumor endothelial cells, which is much larger than gaps between healthy endothelial cells. This difference allows for nanoparticle targeting of tumor tissue.

Nanoparticles of a diameter of greater than 40 nm cannot enter normal tissue because the gap between healthy endothelial cells is too small. However, nanoparticles of a diameter of between 60 and 400 nm selectively penetrate tumor tissue regions because the gap between individual tumor endothelial cells is large enough to allow for entry. Nanoparticles large enough for selective tumor tissue penetration (diameter of between about 60 and about 400 nm) cannot be localized internally by tumor cells through pinocytosis, which is limited to particles of a diameter of less than about 20 nm. Accordingly, nanoparticles which can selectively penetrate tumor tissue release the chemotherapeutic payload extracellularly through degradation of the polymer matrix, which results in unselective delivery of the active ingredient.

Thus, what is needed are nanoparticles large enough for selective tumor tissue penetration (diameter of between 60 and 400 nm) which can decompose within the tissue to a size amenable for cellular uptake (diameter of less than about 20 nm) by tumor cells. The above concept may have broad applicability beyond selective delivery of active ingredients to tumor tissues.

SUMMARY

The present invention satisfies these and other needs by providing in one aspect, a composite nanoparticle. The composite nanoparticle includes subunit nanoparticles of between about 10 nm and about 20 nm diameter which contain an active ingredient, wherein the composite nanoparticle has a diameter of between about 60 nm and about 400 nm.

In another aspect, a method for forming composite nanoparticles is provided. Subunit nanoparticles of diameter between about 10 nm and about 20 nm which include an active ingredient are synthesized and assembled into composite nanoparticles of diameter between about 60 nm and about 400 nm.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

“Alkyl” by itself or as part of another substituent, refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne. Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-l-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. The term “alkyl” is specifically intended to include groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used. In some embodiments, an alkyl group comprises from 1 to 20 carbon atoms (C₁-C₂₀ alkyl). In other embodiments, an alkyl group comprises from 1 to 10 carbon atoms (C₁-C₁₀ alkyl). In still other embodiments, an alkyl group comprises from 1 to 6 carbon atoms (C₁-C₆ alkyl).

“Alkanyl” by itself or as part of another substituent, refers to a saturated branched, straight-chain or cyclic alkyl radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl” by itself or as part of another substituent, refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl , but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Acyl” by itself or as part of another substituent refers to a radical —C(O)R⁴⁰⁰, where R⁴⁰⁰ is hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroarylalkyl or substituted heteroarylalkyl as defined herein. Representative examples include, but are not limited to formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.

“Antibody” as used herein refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes, e.g., a fragment containing one or more complementarity determining region (CDR). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are typically classified as either, e.g., kappa or lambda. Heavy chains are typically classified e.g., as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody) structural unit comprises a tetramer. In nature, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2 (fragment antigen binding) and Fc (fragment crystallizable, or fragment complement binding). F(ab)′2 is a dimer of Fab, which itself is a light chain joined to VH-CH by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab′)2 dimer into a Fab′ monomer. The Fab′ monomer is essentially a Fab with part of the hinge region. The Fc portion of the antibody molecule corresponds largely to the constant region of the immunoglobulin heavy chain, and is responsible for the antibody's effector function (see, Fundamental Immunology, 4^(th) edition. W. E. Paul, ed., Raven Press, N.Y. (1998), for a more detailed description of antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab′ or Fc fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology, peptide display, or the like. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibodies also include single-armed composite monoclonal antibodies, single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, as well as diabodies, tribodies, and tetrabodies (Pack et al., (1995) J Mol Blot 246:28; Biotechnol 11:1271; and Biochemistry 31:1579). The antibodies are, e.g., polyclonal, monoclonal, chimeric humanized, single chain, Fab fragments, fragments produced by a Fab expression library, or the like.

“Aryl” by itself or as part of another substituent, refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system, as defined herein. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like. In some embodiments, an aryl group comprises from 6 to 20 carbon atoms (C₆-C₂₀ aryl). In other embodiments, an aryl group comprises from 6 to 15 carbon atoms (C₆-C₁₅ aryl). In still other embodiments, an aryl group comprises from 6 to 15 carbon atoms (C₆-C₁₀ aryl).

“Arylalkyl” by itself or as part of another substituent, refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp^(a) carbon atom, is replaced with an aryl group as, as defined herein. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is used. In some embodiments, an arylalkyl group is (C₆-C₃₀) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₁₀) alkyl and the aryl moiety is (C₆-C₂₀) aryl. In other embodiments, an arylalkyl group is (C₆-C₂₀) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₈) alkyl and the aryl moiety is (C₆-C₁₂) aryl. In still other embodiments, an arylalkyl group is (C₆-C₁₅) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₅) alkyl and the aryl moiety is (C₆-C₁₀) aryl.

“Compounds” refers to compounds encompassed by structural formulae disclosed herein and includes any specific compounds within these formulae whose structure is disclosed herein. Compounds may be identified either by their chemical structure and/or chemical name. When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound. The compounds described herein may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The compounds may also exist in several tautomeric forms including the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds. The compounds described also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds described herein include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, etc. In general, it should be understood that all isotopes of any of the elements comprising the compounds described herein may be found in these compounds. Compounds may exist in unsolvated or unhydrated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, compounds may be hydrated, solvated or N-oxides. Certain compounds may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present invention. Further, it should be understood, when partial structures of the compounds are illustrated, that brackets indicate the point of attachment of the partial structure to the rest of the molecule.

“Heteroalkyl,” “Heteroalkanyl,” “Heteroalkenyl” and “Heteroalkynyl” by themselves or as part of other substituents, refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and optionally any associated hydrogen atoms), are each, independently of one another, replaced with the same or different heteroatoms or heteroatomic groups. Typical heteroatoms or heteroatomic groups which can replace the carbon atoms include, but are not limited to, —O—, —S—, —N—, —Si—, —NH—, —S(O)—, —S(O)₂—, —S(O)NH—, —S(O)₂NH— and the like and combinations thereof. The heteroatoms or heteroatomic groups may be placed at any interior position of the alkyl, alkenyl or alkynyl groups. Typical heteroatomic groups which can be included in these groups include, but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR⁵⁰¹R⁵⁰²—, ═N—N═, —N═N—, —N═N—NR⁵⁰³R⁵⁰⁴, —PR⁵⁰⁵—, —P(O)₂—, —POR⁵⁰⁶—, —O—P(O)₂—, —SO—, —SO₂—, —SnR⁵⁰⁷R⁵⁰⁸— and the like, wherein R⁵⁰¹, R⁵⁰², R⁵⁰³, R⁵⁰⁴, R⁵⁰⁵, R⁵⁰⁶, R⁵⁰⁷ and R⁵⁰⁸ are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.

“Heteroaryl” by itself or as part of another substituent, refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring systems, as defined herein. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. In some embodiments, the heteroaryl group comprises from 5 to 20 ring atoms (5-20 membered heteroaryl). In other embodiments, the heteroaryl group comprises from 5 to 10 ring atoms (5-10 membered heteroaryl). Exemplary heteroaryl groups include those derived from furan, thiophene, pyrrole, benzothiophene, benzofuran, benzimidazole, indole, pyridine, pyrazole, quinoline, imidazole, oxazole, isoxazole and pyrazine.

“Heteroarylalkyl” by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp^(a) carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylakenyl and/or heteroarylalkynyl is used. In some embodiments, the heteroarylalkyl group is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is (C₁-C₆) alkyl and the heteroaryl moiety is a 5-15-membered heteroaryl. In other embodiments, the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C₁-C₃) alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

“Hydrates” refers to incorporation of water into to the crystal lattice of a compound described herein, in stoichiometric proportions, resulting in the formation of an adduct. Methods of making hydrates include, but are not limited to, storage in an atmosphere containing water vapor, dosage forms that include water, or routine pharmaceutical processing steps such as, for example, crystallization (i.e., from water or mixed aqueous solvents), lyophilization, wet granulation, aqueous film coating, or spray drying. Hydrates may also be formed, under certain circumstances, from crystalline solvates upon exposure to water vapor, or upon suspension of the anhydrous material in water. Hydrates may also crystallize in more than one form resulting in hydrate polymorphism. See e.g., (Guillory, K., Chapter 5, pp. 202-205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, N.Y., 1999). The above methods for preparing hydrates are well within the ambit of those of skill in the art, are completely conventional and do not require any experimentation beyond what is typical in the art. Hydrates may be characterized and/or analyzed by methods well known to those of skill in the art such as, for example, single crystal X-ray diffraction, X-ray powder diffraction, Polarizing optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In addition, many commercial companies routine offer services that include preparation and/or characterization of hydrates such as, for example, HOLODIAG, Pharmaparc II, Voie de I′Innovation, 27 100 Val de Reuil, France (http://www.holodiag.com).

“Parent Aromatic Ring System” refers to an unsaturated cyclic or polycyclic ring system having a conjugated π electron system. Specifically included within the definition of “parent aromatic ring system” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, etc. Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like.

“Ligand” as used herein refers to an oligonucleotide, single stranded RNA, single stranded DNA, a DNA binding protein, a RNA binding protein, a peptide nucleic acid, a peptide, a depsipeptide, a polypeptide, an antibody, a peptoid, a polymer, a polysiloxane, an inorganic compound of molecular weight greater that 50 daltons, an organic compound of molecular weight between about 1000 daltons and about 50 daltons or a combination thereof.

“Peptide” as used herein refers to a polymer of amino acid residues between about 2 and 50 amino acid residues, between about 2 and 20 amino acid residues, or between about 2 and 10 residues. Peptides include modified peptides such as, for example, glycopeptides, PEGylated peptides, lipopeptides, peptides conjugated with organic or inorganic ligands, peptides which contain peptide bond isosteres (e.g., ψ[CH₂S], ψ[CH₂NH₂], ψ[NHCO], ψ[COCH₂], ψ[(E) or (Z) CH═CH], etc. and also include cyclic peptides. In some embodiments, the amino acid residues may be any L-α-amino acid, D-α-amino residue, N-alkyl variants thereof or combinations thereof. In other embodiments, the amino acid residues may any L-α-amino acid, D-α-amino residue, β-amino acids, γ-amino acids, N-alkyl variants thereof or combinations thereof.

“Preventing” or “prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). In some embodiments, “preventing” or “prevention” refers to reducing symptoms of the disease by taking the compound in a preventative fashion. The application of a therapeutic for preventing or prevention of a disease of disorder is known as ‘prophylaxis.’ In some embodiments, the compounds provided herein provide superior prophylaxis because of lower long term side effects over long time periods.

“Prodrug” refers to a derivative of a drug molecule that requires a transformation within the body to release the active drug. Prodrugs are frequently (though not necessarily) pharmacologically inactive until converted to the parent drug.

“Promoiety” refers to a form of protecting group that when used to mask a functional group within a drug molecule converts the drug into a prodrug. Typically, the promoiety will be attached to the drug via bond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

“Recognition Compound” as used herein refers to an oligonucleotide, single stranded RNA, single stranded DNA, a DNA binding protein, a RNA binding protein, a peptide nucleic acid, a peptide, a depsipeptide, a polypeptide, an antibody, a peptoid, a polymer, a polysiloxanes, an inorganic compounds of molecular weight greater that 50 daltons, an organic compounds of molecular weight between about 1000 daltons and about 50 daltons or a combination thereof

“Salt” refers to a salt of a compound, which possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. In some embodiments, the salt is pharmaceutically acceptable.

“Solvates” refers to incorporation of solvents into to the crystal lattice of a compound described herein, in stoichiometric proportions, resulting in the formation of an adduct. Methods of making solvates include, but are not limited to, storage in an atmosphere containing a solvent, dosage forms that include the solvent, or routine pharmaceutical processing steps such as, for example, crystallization (i.e., from solvent or mixed solvents) vapor diffusion, etc. Solvates may also be formed, under certain circumstances, from other crystalline solvates or hydrates upon exposure to the solvent or upon suspension material in solvent. Solvates may crystallize in more than one form resulting in solvate polymorphism. See e.g., (Guillory, K., Chapter 5, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, N.Y., 1999)). The above methods for preparing solvates are well within the ambit of those of skill in the art, are completely conventional do not require any experimentation beyond what is typical in the art. Solvates may be characterized and/or analyzed by methods well known to those of skill in the art such as, for example, single crystal X-ray diffraction, X-ray powder diffraction, Polarizing optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In addition, many commercial companies routine offer services that include preparation and/or characterization of solvates such as, for example, HOLODIAG, Pharmaparc II, Voie de l'Innovation, 27 100 Val de Reuil, France (http://www.holodiag.com).

“Substituted” when used to modify a specified group or radical, means that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent(s). Substituent groups useful for substituting saturated carbon atoms in the specified group or radical include, but are not limited to —R^(a), halo, —O⁻, ═O, —OR^(b), —SR^(b), —S⁻, ═S, —NR^(c)R^(c), ′NR^(b), ═N—OR^(b), trihalomethyl, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R^(b), —S(O)₂NR^(b), —S(O)₂O⁻, —S(O)₂OR^(b), —OS(O)₂R, —OS(O)₂O⁻, —OS(O)₂OR^(b), —P(O)(O⁻)₂, —P(O)(OR^(b))(O⁻), —P(O)(OR^(b))(OR^(b)), —C(O)R^(b), —C(S)R^(b), —C(NR^(b))R^(b), —C(O)O⁻, —C(O)OR^(b), —C(S)OR^(b), —C(O)NR^(c)R^(c), —C(NR^(b))NR^(c)R^(c), —OC(O)R^(b), —OC(S)R^(b), —OC(O)O⁻, —OC(O)OR^(b), —OC(S)OR^(b), —NR^(b)C(O)R^(b), —NR^(b)C(S)R^(b), —NR^(b)C(O)O⁻, —NR^(b)C(O)OR^(b), —NR^(b)C(S)OR^(b), —NR^(b)C(O)NR^(c)R^(c), —NR^(b)C(NR^(b))R^(b) and —NR^(b)C(NR^(b))NR^(c)R^(c), where R^(a) is selected from the group consisting of alkyl, cycloalkyl, heteroalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl; each R^(b) is independently hydrogen or R^(a); and each R^(c) is independently R^(b) or alternatively, the two R^(c)'s are taken together with the nitrogen atom to which they are bonded form a 4-, 5-, 6- or 7-membered cycloheteroalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S. As specific examples, —NR^(c)R^(c) is meant to include —NH₂, —NH-alkyl, N-pyrrolidinyl and N-morpholinyl.

Similarly, substituent groups useful for substituting unsaturated carbon atoms in the specified group or radical include, but are not limited to, —R^(a), halo, —O⁻, —OR^(b), —SR^(b), —S⁻, —NR^(c)R^(c), trihalomethyl, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)₂R^(b), —S(O)₂O⁻, —S(O)₂OR^(b), —OS(O)₂R^(b), —OS(O)₂O⁻, —OS(O)₂OR^(b), —P(O)(O⁻)₂, —P(O)(OR^(b))(O⁻), —P(O)(OR^(b))(OR^(b)), —C(O)R^(b), —C(S)R^(b), —C(NR^(b))R^(b), —C(O)O⁻, —C(O)OR^(b), —C(S)OR^(b), —C(O)NR^(c)R^(c), —C(NR^(b))NR^(c)R^(c), —OC(O)R^(b), —OC(S)R^(b), —OC(O)O⁻, —OC(O)OR^(b), —OC(S)OR^(b), —NR^(b)C(O)R^(b), —NR^(b)C(S)R^(b), —NR^(b)C(O)O⁻, —NR^(b)C(O)OR^(b), —NR^(b)C(S)OR^(b), —NR^(b)C(O)NR^(c)R^(c), —NR^(b)C(NR^(b))R^(b) and —NR^(b)C(NR^(b))NR^(c)R^(c), where R^(a), R^(b) and R^(c) are as previously defined.

Substituent groups useful for submitting nitrogen atoms in heteroalkyl and cycloheteroalkyl groups include, but are not limited to, —R^(a), —O⁻, —OR^(b), —SR^(b), —S⁻, —NR^(c)R^(c), trihalomethyl, —CF₃, —CN, —NO, —NO₂, —S(O)₂R^(b), —S(O)₂O⁻, —S(O)₂OR^(b), —OS(O)₂R^(b), —OS(O)₂O⁻, —OS(O)₂OR^(b), —P(O)(O⁻)₂, —P(O)(OR^(b))(O⁻), —P(O)(OR^(b))(OR^(b)), —C(O)R^(b), —C(S)R^(b), —C(NR^(b))R^(b), —C(O)OR^(b), —C(S)OR^(b), —C(O)NR^(c)R^(c), —C(NR^(b))NR^(c)R^(c), —OC(O)R^(b), —OC(S)R^(b), —OC(O)OR^(b), —OC(S)OR^(b), —NR^(b)C(O)R^(b), —NR^(b)C(S)R^(b), —NR^(b)C(O)OR^(b), —NR^(b)C(S)OR^(b), —NR^(b)C(O)NR^(c)R^(c), —NR^(b)C(NR^(b))R^(b) and —NR^(b)C(NR^(b))NR^(c)R^(c), where R^(a), R^(b) and R^(c) are as previously defined.

Substituent groups from the above lists useful for substituting other specified groups or atoms will be apparent to those of skill in the art. The substituents used to substitute a specified group can be further substituted, typically with one or more of the same or different groups selected from the various groups specified above. In some embodiments, substituents are limited to the groups above.

“Subject,” “individual” or “patient” is used interchangeably herein and refers to a vertebrate, preferably a mammal. Mammals include, but are not limited to, murines, rodents, simians, humans, farm animals, sport animals and pets.

“Treating” or “treatment” of any disease or disorder refers, in some embodiments, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof,). Treatment may also be considered to include preemptive or prophylactic administration to ameliorate, arrest or prevent the development of the disease or at least one of the clinical symptoms. Treatment can also refer to the lessening of the severity and/or the duration of one or more symptoms of a disease or disorder.

In a further feature, the treatment rendered has lower potential for long term side effects over multiple years. In other embodiments “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet other embodiments, “treating” or “treatment” refers to inhibiting the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter) or both. In yet other embodiments, “treating” or “treatment” refers to delaying the onset of the disease or disorder.

“Therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, adsorption, distribution, metabolism and excretion etc., of the patient to be treated.

“Vehicle” refers to a diluent, excipient or carrier with which a compound is administered to a subject. In some embodiments, the vehicle is pharmaceutically acceptable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of Type A prodrug II and Type B Prodrug II.

FIG. 2 is an illustration of a microphase separated structure.

DETAILED DESCRIPTION Composite Nanoparticles, Subunit Nanoparticles and Methods of Synthesis Thereof

Provided herein are composite nanoparticles which include subunit nanoparticles including at least one active ingredient. In some embodiments, the subunit nanoparticles include more than one active ingredient. In other embodiments, the active ingredient is a drug or prodrug useful for treating, preventing or ameliorating various medical disorders. In still other embodiments, the drug is an anticancer drug. In still other embodiments, the active ingredient is a diagnostic agent.

In some embodiments, composite nanoparticles include subunit nanoparticles of a diameter between about 10 nm and about 20 nm which incorporate at least one active ingredient, where the composite nanoparticles are of a diameter of between about 60 nm and about 400 nm. In other embodiments, composite nanoparticles are of a diameter of between about 100 nm and about 300 nm. In still other embodiments, composite nanoparticles are of a diameter of between about 125 nm and about 200 nm. In still other embodiments, composite nanoparticles are of a diameter of between about 125 nm and about 175 nm.

The physical properties of composite nanoparticles may be modified by coating the surface thereof. In some embodiments, composite nanoparticles are coated with a hydrophilic layer. In other embodiments, the hydrophilic layer is PEG, polyvinylpyrrolidine, polyvinyl alcohol, albumin, polyacrylic acid, poloxamine, lauryl ethers, lipids, proteins, poloxamers, polysorbate, vitamin E TPGS, polysaccharides, copolymers or combinations thereof.

Composite nanoparticles may be specifically targeted to particular tissues with an organism. Accordingly, in some embodiments, composite nanoparticles are coated with recognition elements. Recognition elements include antibodies, ligands, carbohydrates or nucleic acids. Ligands which are recognized by the recognition elements include, but are not limited to, folate, biotin, tarquidar, RGD peptide or RGD peptidomimetic.

In some embodiments subunit nanoparticles include dendrimers or block polymers. Block polymers include, but are not limited to, block polymers used to form prodrugs of drugs, infra. Dendrimers include, but are not limited to, commercially available dendrimers such as PANAM dendrimers, PEG-core dendrimers, phosphorus dendrimers or polypropyenamine dendrimers, which are commercially available from suppliers such as Sigma Aldrich (St. Louis, Mo.) and Dendritech, Inc. (Midland, Mich. 48642).

In general, composite nanoparticles are synthesized by providing subunit nanoparticles of diameter between about 10 nm and about 20 nm, which include a drug. The subunit nanoparticles are then assembled into composite nanoparticles of diameter between about 60 nm and about 400 nm.

In some embodiments, subunit nanoparticles are crosslinked to form a composite nanoparticle. A number of conceptual methods exist to crosslink subunit nanoparticles to form composite nanoparticles. In some embodiments, a polymeric prodrug is used to form subunit nanoparticles which are then crosslinked by addition of a linker, infra. In other embodiments, a linker is added to a polymeric prodrug prior to forming subunit nanoparticles and crosslinking takes place during composite nanoparticle formation. In still other embodiments, nanoparticles are formed from a mixture of drug and/or prodrug and a carrier polymer and the carrier polymer is crosslinked via a linker to form a composite nanoparticle. In still other embodiments, composite nanoparticles are formed from a mixture of drug and/or prodrug, carrier polymer and a linker which form subunit nanoparticles which are then crosslinked in situ. In still other embodiments, subunit nanoparticles may be formed into a composite liposomal nanoparticle for example, the methods disclosed in “Liposome Technology, Volume I: Liposome Preparation and Related Techniques, Third Edition” (CRC Press, Sep. 12, 2006; edited by Gregory Gregoriadis).

Composite nanoparticles may be crosslinked, for example, by amide, ester, carbamate, hydrazone, acetal, cis-aconityl, urea, sulfonamide, disulfide, carbon-carbon or sulfonyl urea bond formation. Many polymers, which include functional groups useful for crosslinking via such bond formation are commercially available and are well known to the skilled artisan.

In other implementations, crosslinked nanoparticles may be formed through ionic attraction. Nanoparticles, which include cationic or anionic groups are formed and a polymer which includes a complementary charged group (i.e., if nanoparticle is cationic, polymer is anionic and vice-versa) is deposited on the surface of the nanoparticle. In this method crosslinking occurs on the nanoparticle surface through formation of ion pairs.

In some embodiments, sustained release formulations may be used to form crosslinked nanoparticles. Here, the decomposition/dissolution of nanoparticles (e.g., by hydrolysis) is primarily controlled not only by polymer composition, but also crosslinking density and/or crosslinking chemistry. In theory, the subunit nanoparticles as well as the composite nanoparticles can be crosslinked.

It should be noted that any of the above methods used to prepare crosslinked composite nanoparticles can also be used to prepare crosslinked subunit nanoparticles.

It is well known in the art that some block polymers, such as, for example, poly(styrene)-b-poly(DL lactide) diblock copolymer can spontaneously self-assemble into microphase separated structures in the melt state, such as that illustrated in FIG. 2. In some embodiments, a type II prodrug, infra, could include a diblock polymer which includes a hydrophobic block connected to a hydrophilic block (e.g., poly(styrene)-b-poly(DL lactide) diblock copolymer to provide microphase separation and form composite and/or subunit nanoparticles. In other embodiments, a drug or prodrug thereof can be blended (no covalent attachment) with a polymer known to exhibit microphase separation to form composite and/or subunit nanoparticles.

Composite nanoparticles may also be made by various conventional methods such as those provided below. In some embodiments, subunit nanoparticles are dispersed in water and added to an agitated water immiscible organic solvent which includes a carrier polymer to form a water in oil solution. The water-in-oil solution is added to an excess of water which includes an emulsifier and the solvent is then removed to provide the composite nanoparticle. In other embodiments, the subunit nanoparticles are dissolved in a water immiscible volatile organic solvent which includes a carrier polymer to form a solution. The water-in-oil solution is added to an excess of water which includes an emulsifier and the solvent is then removed to provide the composite nanoparticle.

In some embodiments, subunit nanoparticles may be made by dissolving an active ingredient in aqueous solution which is added to an agitated organic polymer solution to form a water-in-oil emulsion. The water-in-oil emulsion is added to an excess of water which includes an emulsifier with agitation to form a water-in-oil-in-water emulsion. The organic solvent is then removed to provide the subunit nanoparticle.

In other embodiments, subunit nanoparticles are made by dissolving an active ingredient in volatile water-miscible solvent to form a solution which is then added to an agitated aqueous solution of carrier polymer to form an oil-in-water emulsion. The oil-in-water solution is diluted with water and the solution filtered to collect the nanoparticles.

In still other embodiments, subunit nanoparticles are made by dissolving an active ingredient in a water immiscible volatile organic solvent which includes a carrier polymer to form a solution, which is added to an excess of water which includes an emulsifier and removing the solvent.

In still other embodiments, subunit nanoparticles are made by dissolving an active ingredient in a water miscible solvent and adding the to an excess of water, thus precipitating the subunit nanoparticles which may be collected by filtration.

In still other embodiments, subunit nanoparticles are made by grafting a hydrophobic active ingredient to a water soluble polymer and aggregating the modified polymer. Examples of water soluble polymers, which spontaneously aggregate when attached to hydrophobic active ingredients include hyaluronic acid or inulin.

Synthetic biogradable polyesters such as polylactide, polyglycolide, poly(lactide-co-glycolide), polyhydroxybutyrate, polycaprolactone, and polydioxanone can be used to make subunit anaoparticles and/or composite nanoparticles. The above polymers are relatively hydrophobic (i.e., water insoluble) and can be used to produce subunit nanoparticles which may or may not be conjugated with drug and/or prodrug. The above polymers can also be used as carrier polymers to form composite nanoparticles, and to form composite nanoparticles by block polymer microphase separation methods.

Natural hydrophilic polysaccharides such as hyaluronic acid and inulin are useful for producing subunit nanoparticles and/or composite nanoparticles. The above polymers are relatively hydrophilic (i.e., water soluble) and therefore, must be made slightly hydrophobic (i.e., water insoluble yet dispersible) by conjugating with hydrophobic drug and/or prodrug, and/or other hydrophobic compounds such as those used to make prodrugs in order to be useful in nanoparticle synthesis.

Another approach to preparing sustained release formulations is formation of controlled gelling compositions (i.e., swelling) by modifying hydrophilic polymers such as, for example, hyaluronic acid and inulin mentioned above by conjugating with hydrophobic drugs and/or prodrugs, and/or other hydrophobic compounds to carefully control the hydrophobicity (i.e., water solubility) of the new polymer (the gelling characteristics of nanoparticles is sensitive to the hydrophobicity/hydrophilicity of polymers comprising nanoparticles.) The resulting polymers can also be used as a carrier polymer for the composite nanoparticles.

Carrier polymers include, but are not limited to the block polymers used to form prodrugs of drugs, infra, proteins: albumin, collagen, gelatin, keratin, lectin, zein, polysaccharides, agarose, alginate, carrageenans, cyclodextrins, guar gum, hyaluronic acid, inulin, pectin, starch, xanthan gum, polyanhydrides: poly(adipic acid), poly(sebatic acid), poly(terphthalic acid), phosphorus compounds, polyphosphazenes, polyorthoesters, cellulose derivatives, carboxy methyl cellulose, ethyl cellulose, hydroxy ethyl cellulose, hydroxy propyl cellulose, hydroxy propyl methyl cellulose, methyl cellulose, poly(D-lactide), poly(L-lactide), poly(D,L-lactide), poly(lactide-co-glycolide), polycaprolactone, poly(lactide-co-caprolactone), poly(glycolide-co-caprolactone), branched or linear polyethylenimie, chitosan, poly(N,N′-dimethylaminoethyl methacrylate), poly(-vinyl pyridine), poly(L-lysine), poly(L-histidine) or poly(B-amino ester, poly(l,6-bis-p-carboxyphenoxyhexane-co-sebacic anhydride), pluronic series of poly(ethylene oxide)-polypropylene oxide)-poly(ethylene oxide) block copolymers, hydroxymethyl cellulose, Tween series of polyethoxylated sorbitan esters and sodium dodecyl sulfate and combinations thereof.

Examples of water immiscible solvent include chloroform, methylene chloride, toluene, hexane, ether, etc. Examples of partially water miscible organic solvent include methyl acetate, ethyl acetate, methyl ethyl ketone, propylene carbonate, butyl lactate, isovaleric acid, benzyl alcohol, etc. Examples of completely water miscible solvents include acetone, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, methanol, ethanol, dimethylformamide.

In some embodiments, composite nanoparticles decompose in tumor tissue. In other embodiments, composite nanoparticles decompose below about at the pH of the interstitial space of the tumor tissue, wherein the pH of the cancer interstitial space is between 5.6 and 6.4.

In some of these embodiments the composite nanoparticle included acid sensitive crosslinks such as hydrazone, acetal, cis-aconityl linkages. In other of these embodiments, the composite nanoparticles may include cationic polymers, such as, for example, branched or linear polyethylenimie, chitosan poly (N,N-dimethylaminoethyl methacrylate) poly(4-vinyl pyridine), poly(L-lysine), poly(L-histidine) or poly(B-amino ester).

In still other embodiments, composite nanoparticles decompose enzymatically (e.g., esterases, peptidases, etc.) or under basic conditions (nucleophilic attack).

Drugs and Compounds Used with Composite Nanoparticles

In some embodiments, compounds of Formula (I) are incorporated in the composite nanoparticles described herein:

or salts, hydrates or solvates thereof wherein:

R₁ is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, —SiR₆R₇R₈, alkyl substituted with one or more SiR₆R₇R₈, —CH═NOR₁₃ or optionally R₁ and R₂ together with the atom to which they are connected form a 5, 6 or 7 membered cycloalkyl or cycloheteroalkyl ring or substituted cycloalkyl or cycloheteroalkyl ring;

R2 is hydrogen, alkyl, substituted alkyl, —NO₂ or —NH₂;

R3 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, or —OR₉ or optionally R₃ and R₄ together with the atom to which they are connected form a 5, 6 or 7 membered cycloalkyl or cycloheteroalkyl ring or substituted cycloalkyl or cycloheteroalkyl ring

R₄ is hydrogen or halogen;

R₅ is alkyl, aryl, arylalkyl, acyl or —(L)_(n)—P;

R₆, R₇ or R₈ are independently alkyl or arylalkyl;

R9 is hydrogen, alkyl, substituted alkyl, —C(O)R₁₀—C(O)OR₁₀ or —C(O)NR₁₁R₁₂, —P or —(L)_(n)—P;

R₁₀ is alkyl, substituted alkyl, arylalkyl, substituted alkyl, heteroalkyl or substituted heteroalkyl;

R₁₁, R₁₂ and R₁₃ are independently alkyl, substituted alkyl, arylalkyl, substituted alkyl, heteroalkyl or substituted heteroalkyl or optionally R₁₂ and R₁₃ together with the atom to which they are connected form a 5, 6 or 7 membered cycloheteroalkyl ring or substituted cycloheteroalkyl ring;

L is a linker;

P is a polymer; and

n is 0 or 1

In some embodiments, a compound having the structure of Formula (II) is provided:

In other embodiments, a compound having the structure of Formula (III) is provided:

In still other embodiments, a compound having the structure of Formula (IV) is provided:

In still other embodiments, a compound having the structure of Formula (V) is provided:

In still other embodiments, a compound having the structure of Formula (VI) is provided:

In still other embodiments, a compound having the structure of Formula (VII) is provided:

In still other embodiments, a compound having the structure of Formula (VIII) is provided:

In still other embodiments, a compound having the structure of Formula (IX) is provided:

In still other embodiments, a compound having the structure of Formula (X) is provided:

In still other embodiments, a compound having the structure of Formula (XI) is provided:

In still other embodiments, a compound having the structure of Formula (XII) is provided:

In still other embodiments, a compound having the structure of Formula (XIII) is provided:

In still other embodiments, a compound having the structure of Formula (XIV) is provided:

In some embodiments, R₁, R₂, R₃ and R₄ are not hydrogen. In other embodiments, R₅ is alkyl or acyl. In still other embodiments, R₅ is (C₁₀-C₂₀) alkyl. In still other embodiments, R₅ is n-C₁₀H₂₁, n-C₁₁H₂₃, n-C₁₀H₂₁, n-C₁₂H₂₅, n-C₁₃H₂₇, n-C₁₄H₂₉, n-C₁₅H₃₁, n-C₁₆H₃₃, n-C₁₇H₃₅, n-C₁₈H₃₇, n-C₁₉H₃₉ or n-C₂₀H₄₁. In still other embodiments, R₅ is (C₁₀-C₂₀) acyl. In still other embodiments, R₅ is —C(O)n-C₁₀H₂₁, —C(O)n-C₁₁H₂₃, —C(O)n-C₁₀H₂₁, —C(O)n-C₁₂H₂₅, —C(O)n-C₁₃H₂₇, —C(O)n-C₁₄H₂₉, —C(O)n-C₁₅H₃₁, —C(O)n-C₁₆H₃₃, —C(O)n-C₁₇H₃₅, —C(O)n-C₁₈H₃₇, —C(O)n-C₁₉H₃₉ or —C(O)n-C₁₉H₃₉.

Other compounds which can be incorporated in the composite nanoparticles described herein include, but are not limited to, Ganciclovir, Saquinavir, Lopinavir, Nelfinavir, Amphotericin B, Neostatin, Itraconazole, Ketoconazole, Miconazole, Oxiconazole, Sertaconazole, Griseofulvin, Ciprofloxacin, Moxifloxacin, Ofloxacin, Methoxyfloxacin, Pefloxacin, Norfloxacin, Sparfloxacin, Temafloxacin, Levofloxacin, Lomefloxacin, Cinoxacin, Cloxacillin, Benzylpeniciilin, Phenylmethoxypenicillin, Erythromycin, Rifampicin, Rifapentin, Ibuprofen, Ketoprofen, Naproxen, Cyclosporin A, Pacitaxel (Taxol), Docetaxel (Taxotere), Etoposide (Vp16, Vepesid), Cisplatin(platinol), 5-Fluorouracil, Rapamycin, Doxorubicin, Daunorubicin, ldarubicin, Epirubicin, Capecitabine, Mitomycin C, Amsacrine, Busulfan, Etoposide, Chlorambucil, Chlormethine, Melphalan, Gemcitabine, Benzoylphenylurea, Cyclopamine, Aciclovir, lndinavir, Lamivudine, Stavudine, Nevirapine, Ritonavir, Oxaprozin, Piroxicam, Sulindac, Curcumin, Curcuminoids, Indomethacin, Dexamethasone, Bethamethasone, Cortisone, Tretinoin, Nifedipine, Ellipticine, Indotecan, Indomitecan, Carmustine, Etoposide, Teniposide and Nile red (fluorescent probe). Those of skill in the art will appreciate that prodrugs of any the above compounds may also be include into composite and subunit nanoparticles.

A linker (i.e., L), typically contains at least two functional groups, e.g., one functional group that can be used to form a bond with the hydroxyl group of camptothecin or camptothecin derivative of with a functional group of a known drug and another functional group that can form a bond with a polymer. Typically, though not necessarily, the functional group on the linker that is used to form a bond with camptothecin, camptothecin derivative or another known drug is at one end of the linker and the functional group that is used to form a bond with the polymer is at the other end of the linker.

The linker will typically comprise electrophilic functional groups that can react with the nucleophilic hydroxyl group on the camptothecin or camptothecin derivative to form a covalent bond. The linker can comprise nucleophilic functional groups that can react with electrophilic functional groups like carbonyl, halide, or alkoxyl groups on the polymer. The linker can also comprise electrophilic groups that can react with nucleophilic functional groups on the polymer. Any of these bonds may be formed by conventional methods known in the art. The resulting bonds between the linker, camptothecin or camptothecin derivative and the polymer should be biodegradable. As is well known to those of skill in the art, suitable combinations of electrophilic and nucleophilic drugs can be used to form a polymeric prodrug of a known drug.

The linker can be of varying lengths, such as from 1 to 20 atoms in length. For example, the linker moiety can be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms in length, where any of the stated values can form an upper and/or lower end point of a range. Further, the linker moiety can be substituted or unsubstituted. When substituted, the linker can contain substituents attached to the backbone of the linker or substituents embedded in the backbone of the linker. For example, an amine substituted linker moiety can contain an amine group attached to the backbone of the linker or a nitrogen in the backbone of the linker.

Suitable linkers include, but are not limited to alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, ethers, esters, polyesters, polyesters, polyalkylenes, polyamines and the like. In a specific example, the linker can comprise —(CH₂)_(m)— wherein m is from 1 to 20, and where the point of attachment to the camptothecin, camptothecin derivative or other known drug is an ether, ester, carbonate or carbamate bond and the point of attachment to the polymer is an ester, ether, carbamate, amine, or amide bond. For example, the linker moiety can be X¹—(CH₂)_(m)—X², wherein m is from 1 to 10, and X¹ is —C(O)—, a bond or —C(O)N—, —C(O)O— and X² —C(O), —C(O)O—, —C(O)N—, —NH—, —O— or halo.

In still another embodiment, the linker can comprise a branched or straight-chain alkyl, wherein one or more of the carbon atoms is substituted with oxygen (e.g., an ether) or an amino group. For example, suitable linkers can include, but are not limited to, a methoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, propoxymethyl, propoxyethyl, methylaminomethyl, methylaminoethyl, methylaminopropyl, methylaminobutyl, ethylaminomethyl, ethylaminoethyl, ethylaminopropyl, propylaminomethyl, propylaminoethyl, methoxymethoxymethyl, ethoxymethoxymethyl, methoxyethoxymethyl, methoxymethoxyethyl, and the like, and derivatives thereof, where the point of attachment to the hydrophobic drug and/or amino acid is an ester, ether, or amide bond.

Suitable polymers for forming a prodrug of known drugs including camptothecin or camptothecin derivatives, include, but are not limited to poly(D-lactide), poly(L-lactide), poly(D,L-lactide), poly(lactide-co-glycolide), polycaprolactone, poly(lactide-co-caprolactone), poly(glycolide-co-caprolactone), branched or linear polyethylenimie, chitosan, poly(N,N′-dimethylaminoethyl methacrylate), poly(4-vinyl pyridine), poly(L-lysine), poly(L-histidine) or poly(B-amino ester and/or poly(1,6-bis-p-carboxyphenoxyhexane-co-sebacic anhydride. These copolymers may take the form of random, block, or alternating copolymer, depending on the monomer sequence. Other polymers of use include, but are not limited to, PHMPA, PHMPA-PLMA, PEG, dextran, chitosan and pullan. The above polymers can be used as carrier polymers in synthesizing both subunit and composite nanoparticles.

In some of the above embodiments, a prodrug of a drug is incorporated into the composite and/or subunit nanoparticle. In other of the above embodiments, the composite and/or subunit nanoparticle includes a block polymer prodrug of a drug. In still other of the above embodiments, the composite nanoparticle and/or subunit nanoparticle includes an amphiphilic graft polymer of the drug. In still other of the above embodiments, the composite nanoparticle and/or subunit nanoparticle includes a hydrophobic ester of the drug and a block polymer prodrug of the drug. In still other of the above embodiments, the composite nanoparticle and/or subunit nanoparticle includes a prodrug of SN-38 or topetecan. In still other of the above embodiments, the composite nanoparticle and/or subunit nanoparticle includes bis-prodrugs such as type A prodrug II and type B prodrug II such as those illustrated in FIG. 1.

Referring now to FIG. 1, it should be noted that the polymer attached to prodrug I, which is typically a conventional prodrug (i.e., not a polymer) may be a block polymer. Furthermore, it should be noted that type A prodrug II, where prodrug I is attached at a fixed location (e.g., at the end of the polymer chain may also be considered a block polymer because it has a well-defined structure.

Conversely type B prodrug II where the prodrug is attached to polymer at a random is not a block polymer because of the ill-defined structure.

Methods for the preparation of compounds of Formulae (I)-(XIV) and prodrugs of known drugs are conventional and within the ambit of the skilled artisan.

Compositions and Methods of Administration

The compositions provided herein contain therapeutically effective amounts of one or more of the composite nanoparticles provided herein that are useful in the prevention, treatment, or amelioration of one or more of the symptoms of diseases or disorders described herein and a vehicle. Vehicles suitable for administration of the composite nanoparticles provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

In addition, the composite nanoparticles may be formulated as the sole active ingredient in the composition or may be combined with other active ingredients.

The compositions contain one or more composite nanoparticles provided herein. The composite nanoparticles are, in some embodiments, formulated into suitable preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as topical administration, transdermal administration and oral inhalation via nebulizers, pressurized metered dose inhalers and dry powder inhalers. In some embodiments, the composite nanoparticles described above are formulated into compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, Seventh Edition (1999).

In the compositions, effective concentrations of one or more composite nanoparticles are mixed with a suitable vehicle. The composite nanoparticles may be derivatized as the corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, ion-pairs, hydrates or prodrugs prior to formulation, as described above. The concentrations of the composite nanoparticles in the compositions are effective for delivery of an amount, upon administration that treats, leads to prevention, or amelioration of one or more of the symptoms of diseases or disorders described herein. In some embodiments, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of a composite nanoparticle is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated.

The active composite nanoparticle is included in the vehicle in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be predicted empirically by testing the composite nanoparticles in in vitro and in vivo systems well known to those of skill in the art and then extrapolated therefrom for dosages for humans. Human doses are then typically fine-tuned in clinical trials and titrated to response.

The concentration of active composite nanoparticles in the composition will depend on absorption, inactivation and excretion rates of the active composite nanoparticle, the physicochemical characteristics of the composite nanoparticle, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to ameliorate one or more of the symptoms of diseases or disorders as described herein.

In some embodiments, a therapeutically effective dosage should produce a serum concentration of composite nanoparticle of from about 0.001 ng/ml to about 1.0 ng/ml, 2-10 ng/ml, 11 to 50 ng/ml, 51-200 ng/ml, or about 200 to 1000 ng/ml. The compositions, in other embodiments, should provide a dosage of from about 0.0001 mg to about 70 mg of composite nanoparticle per kilogram of body weight per day. Dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, or about 1000 mg, and in some embodiments from about 10 mg to about 500 mg of the composite nanoparticle per dosage unit form.

The composite nanoparticle may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data or subsequent clinical testing. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

In instances in which the composite nanoparticles exhibit insufficient solubility, methods for solubilizing composite nanoparticles may be used such as use of liposomes or emulsions. Such methods are known to those of skill in this art, and include, but are not limited to, using co-solvents, such as dimethylsulfoxide (DMSO), using surfactants or surface modifiers, such as TWEEN®, complexing agents such as cyclodextrin or dissolution by enhanced ionization (i.e. dissolving in aqueous sodium bicarbonate).

Upon mixing or addition of the composite nanoparticle(s), the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the composite nanoparticle in the selected vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.

The compositions are provided for administration to humans and animals in indication appropriate dosage forms, such as dry powder inhalers (DPIs), pressurized metered dose inhalers (pMDIs), nebulizers, tablets, capsules, pills, sublingual tapes/bioerodible strips, tablets or capsules, powders, granules, lozenges, lotions, salves, suppositories, fast melts, transdermal patches or other transdermal application devices/preparations, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the composite nanoparticles. The therapeutically active composite nanoparticles are, in some embodiments, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refer to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active composite nanoparticle sufficient to produce the desired therapeutic effect, in association with the required vehicle. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

Liquid compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional adjuvants in a vehicle, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension, colloidal dispersion, emulsion or liposomal formulation. If desired, the composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.

Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975 or later editions thereof.

Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from vehicle or carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001%-100% active ingredient, in one embodiment 0.1-95%, in another embodiment 0.4-10%.

In certain embodiments, the compositions are lactose-free compositions containing excipients that are well known in the art and are listed, for example, in the U.S. Pharmacopeia (USP) 25-NF20 (2002). In general, lactose-free compositions contain active ingredients, a binder/filler, and a lubricant in compatible amounts. Particular lactose-free dosage forms contain active ingredients, microcrystalline cellulose, pre-gelatinized starch, and magnesium stearate.

Further provided are anhydrous compositions and dosage forms comprising active ingredients, since water can facilitate the degradation of some composite nanoparticles. For example, the addition of water (e.g., 5%) is widely accepted as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water and heat accelerate the decomposition of some composite nanoparticles. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.

Anhydrous compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.

An anhydrous composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are generally packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

Oral dosage forms are either solid, gel or liquid. The solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms such as for example, capsules or tablets. The tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or composite nanoparticles of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an enteric coating; a film coating agent and modified release agent. Examples of binders include microcrystalline cellulose, methyl paraben, polyalkyleneoxides, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidones, sucrose and starch and starch derivatives. Lubricants include talc, starch, magnesium/calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, trehalose, lysine, leucine, lecithin, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate and advanced coloring or anti-forgery color/opalescent additives known to those skilled in the art. Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of composite nanoparticles which produce a pleasant sensation or mask unpleasant taste, such as, but not limited to peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.

Enteric-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate. Modified release agents include polymers such as the Eudragit® series and cellulose esters.

The composite nanoparticle, or derivative thereof, can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active composite nanoparticle in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The composite nanoparticles can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup may contain, in addition to the active composite nanoparticles, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is a composite nanoparticle as described herein. Higher concentrations, up to about 98% by weight of the active ingredient may be included.

In all embodiments, tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient. Thus, for example, they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.

Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations. Vehicles used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid. Carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use suspending agents and preservatives. Acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of composite nanoparticles which produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for example, propylene carbonate, vegetable oils or triglycerides, is in some embodiments encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a liquid vehicle, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active composite nanoparticle or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Patent Nos. RE 28,819 and U.S. Pat. No. 4,358,603. Briefly, such formulations include, but are not limited to, those containing a composite nanoparticle provided herein, a dialkylated mono- or polyalkylene glycol, including, but not limited to, 1,2-dimethoxyethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholic solutions including an acetal. Alcohols used in these formulations are any water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di (lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

Parenteral administration, in some embodiments characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

Vehicles used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other substances.

Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (Tween® 80). A sequestering or chelating agent of metal ions includes EDTA. Carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The concentration of composite nanoparticle is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight, body surface area and condition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active composite nanoparticle is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In some embodiments, a therapeutically effective dosage is formulated to contain a concentration of at least about 0.01% w/w up to about 90% w/w or more, in certain embodiments more than 0.1% w/w of the active composite nanoparticle to the treated tissue(s).

The composite nanoparticle may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the composite nanoparticle in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.

Of interest, herein are also lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.

The sterile, lyophilized powder is prepared by dissolving a composite nanoparticle provided herein, or a derivative thereof, in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, an antioxidant, a buffer and a bulking agent. In some embodiments, the excipient is selected from dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose and other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In some embodiments, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the composite nanoparticle. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carriers. The precise amount depends upon the selected composite nanoparticle. Such amount can be empirically determined.

Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.

The composite nanoparticles or derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microtine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, in some embodiments, have mass median geometric diameters of less than 5 microns, in other embodiments less than 10 microns.

The composite nanoparticles may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active composite nanoparticle alone or in combination with other excipients can also be administered.

For nasal administration, the preparation may contain an esterified phosphonate composite nanoparticle dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application. The carrier may contain solubilizing or suspending agents such as propylene glycol, surfactants, absorption enhancers such as lecithin or cyclodextrin, or preservatives.

Solutions, particularly those intended for ophthalmic use, may be formulated as 0.01% - 10% isotonic solutions, pH about 5-7.4, with appropriate salts.

Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, and rectal administration, are also contemplated herein.

Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957.

For example, dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The weight of a rectal suppository, in one embodiment, is about 2 to 3 gm. Tablets and capsules for rectal administration are manufactured using the same substance and by the same methods as for formulations for oral administration.

The composite nanoparticles provided herein, or derivatives thereof, may also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874.

In some embodiments, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Pat. No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) may be formed by drying down phosphatidyl choline and phosphatidyl serine (7:3 molar ratio) on the inside of a flask. A solution of a composite nanoparticle provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed. The resulting vesicles are washed to remove encapsulated composite nanoparticle, pelleted by centrifugation, and then suspended in PBS.

The composite nanoparticles described herein may be formulated in compositions described in the art for delivery of hydrophobic medicines (see, e.g., U.S. Pat. Nos. 5,645,856, 6,096,338, 5,510,103, 5,955,509, 6,322,817, 6,555,139, 6,579,519, 6,746,653, 6,834,791, 7,094,810, 8,715,741 and 8,414,926) or a s composite nanoparticles described in copending U.S. Provisional Patent No. 62/305,519.

The composite nanoparticles or derivatives may be packaged as articles of manufacture containing packaging material, a composite nanoparticle or derivative thereof provided herein, which is effective for treatment, prevention or amelioration of one or more symptoms of the diseases or disorders, supra, within the packaging material, and a label that indicates that the composite nanoparticle or composition or derivative thereof, is used for the treatment, prevention or amelioration of one or more symptoms of the diseases or disorders, supra.

The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging products are well known to those of skill in the art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the composite nanoparticles and compositions provided herein are contemplated as are a variety of treatments for any disease or disorder described herein.

Dosages

In human therapeutics, the physician will determine the dosage regimen that is most appropriate according to a preventive or curative treatment and according to the age, weight, stage of the disease and other factors specific to the subject to be treated. The compositions, in other embodiments, should provide a dosage of from about 0.0001 mg to about 70 mg of composite nanoparticle per kilogram of body weight per day. Dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, or about 1000 mg, and in some embodiments from about 10 mg to about 500 mg of the active ingredient or a combination of essential ingredients per dosage unit form.

The amount of active ingredient in the formulations provided herein, which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof, will vary with the nature and severity of the disease or condition, and the route by which the active ingredient is administered. The frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject.

Exemplary doses of a formulation include milligram or microgram amounts of the active composite nanoparticle per kilogram of subject (e.g., from about 1 micrograms per kilogram to about 50 milligrams per kilogram, from about 10 micrograms per kilogram to about 30 milligrams per kilogram, from about 100 micrograms per kilogram to about 10 milligrams per kilogram, or from about 100 microgram per kilogram to about 5 milligrams per kilogram).

It may be necessary to use dosages of the active ingredient outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response.

Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the composition provided herein are also encompassed by the above described dosage amounts and dose frequency schedules. Further, when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.

In certain embodiments, administration of the same formulation provided herein may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

Methods of Use of the Composite Nanoparticles Compositions

Methods of treating, preventing, or ameliorating one or more symptoms of diseases including, for example, cancer, viral infection, fungal infection, bacterial infection and inflammation also provided herein. The compounds described herein may be used to treat or prevent cancers and/or tumors of the anus, bile duct, bladder, bone, bone marrow, bowel (including colon and rectum), breast, eye, gall bladder, kidney, mouth. larynx, esophagus, stomach, testis, cervix, head, neck, ovary, lung, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, pancreas, prostate, blood cells (including lymphocytes and other immune system cells), and brain. Specific cancers contemplated for treatment include carcinomas, Karposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, and other), and lymphoma (Hodgkin's and non-Hodgkin's), and multiple myeloma.

Other examples of cancers that can be treated according to the methods disclosed herein are adrenocortical carcinoma, adrenocortical carcinoma, cerebellar astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoid tumor, central nervous system lymphoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, retinoblastoma, islet cell carcinoma (endocrine pancreas), laryngeal cancer, lip and oral cavity cancer, liver cancer, medulloblastoma, Merkel cell carcinoma, squamous neck cancer with occult mycosis fungoides, myelodysplastic syndromes, myelogenous leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing's sarcoma, soft tissue sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia, and Wilms' tumor.

In practicing the methods, therapeutically effective amounts of the compounds or compositions, described herein, supra, are administered.

Combination Therapy

The composite nanoparticles and compositions disclosed herein may also be used in combination with one or more other active ingredients. In certain embodiments, the composite nanoparticles may be administered in combination, or sequentially, with another therapeutic agent. Such other therapeutic agents include those known for treatment, prevention or amelioration of one or more symptoms associated with, for example cancer.

It should be understood that any suitable combination of the composite nanoparticles and compositions provided herein with one or more of the above therapeutic agents and optionally one or more further pharmacologically active substances are considered to be within the scope of the present disclosure. In some embodiments, the composite nanoparticles and compositions provided herein are administered prior to or subsequent to the one or more additional active ingredients.

Finally, it should be noted that there are alternative ways of implementing the present invention. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims.

All publications and patents cited herein are incorporated by reference in their entirety.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES Example 1: Preparation of Compound 1

To a solution of SN38 (1 g, 2.55 mmol, 1.0 eq.) in dichloromethane (400 mL) were added pyridine (5 mL) and Boc anhydride (6.39 g, 6.371 mmol, 2.5 eq.) and the reaction mixture was stirred at room temperature for 36 h. The reaction mixture was filtered and washed with 0.01N HCl (3×). The organic phase was separated, dried (sodium sulfate) and evaporated under reduced pressure to provide compound 1 (1.26 g), which was used in the next step without further purification.

Example 2: Preparation of Compound 2

To a solution of compound 1 (630 mg, 1.3 mmol, 1.0 eq) in dichloromethane (20 mL) were added N,N′-dicyclohexylcarbodiimide (DCC) (538 mg, 2.60 mmol, 2.0 eq), DMAP (190 mg, 1.56 mmol, 1.2 eq) and valeric acid (265 mg, 2.6 mmol 2.0 eq) and the reaction mixture was stirred at room temperature for 24 h.

The mixture was then filtered, dried (MgSO4) and solvent was evaporated to provide a residue, which was purified by column chromatography to yield the valerate ester of compound 1. The ester was then treated with 80% TFA in DCM and stirred at room temperature for 3 h. TFA was removed and the crude was purified by column chromatography to provide compound 2(LGP001).

Example 3: Preparation of Compound 3

To a solution of an activated ester of poly(lactic-co-glycolic acid) (PLGA-NHS (100 mg, 0.0066 mmol, 1.0 eq, Akina, Inc., 3495, Kent Ave. West Lafayette, Ind. 47906, cat.# A1096, approximate molecular weight of 15000)) in DMF (4 mL) was added hydroxyl compound 2 (4.71 mg, 0.0099 mmol, 1.5 eq.) and DIEA (0.1 mL). The reaction mixture was stirred at room temperature for 12 h, diluted with ether to precipitate the polymer, placed in the freezer overnight and the solvents were decanted to provide a yellow viscous material. The yellow material was washed with ether (2×) and the flask was placed under vacuum to provide a yellow foamy material.

Example 4: Preparation of 1% Solutions of Polymer Conjugate in Acetone and Dichloromethane

10 mg of polymer conjugate 3 was added to 1 mL of the corresponding solvent and stirred at room temperature to provide a clear homogeneous solution which was directly used in the next step.

Example 5: Preparation of 10% Solutions of Polymer Conjugate in Acetone and Dichloromethane

100 mg of polymer conjugate 3 was added tol mL of the corresponding solvent and stirred at room temperature to provide a clear homogeneous solution which was directly used in the next step.

Example 6: Preparation of 1% aq. Polyvinyl Alcohol Solution.

2 g of Polyvinyl alcohol (PVA) (87-80% hydrolyzed, Alfa Aesar, Haverill, Mass., Cat# 41240) was added to 200 mL of distilled water, heated and stirred until a clear homogenous solution was formed. Then the solution was allowed to slowly cool down using a water bath and used in the next step.

Example 7: Composite Nanoparticle Formation

To a vigorously stirred solution of 1% aq. polyvinyl alcohol solution (20 g) in an open vial, the polymer conjugate solution 3 (1 mL of 1% or 10% in either acetone (water miscible) or dichloromethane (water immiscible) was added dropwise with a syringe. Vigorous stirring was continued at room temperature in an open vial to evaporate the solvent for which required 3 hours for acetone and 24 hours for dichloromethane.

No composite nanoparticles were observed in the 1% acetone solution after stirring for three hours and after centrifugation at 2,000 rpm for two minutes. The three other solutions provided precipitated nanoparticles after stirring and centrifugation at 2,000 rpm for two minutes. With the dichloromethane solutions a probe sonicator was used to first disperse the polymer conjugate 3 as a fine oil-in-water emulsion in polyvinyl alcohol prior to stirring. 

1. A method for forming composite nanoparticles comprising: making subunit nanoparticles of diameter between about 10 nm and about 20 nm which include an active ingredient; and assembling the subunit nanoparticles into composite nanoparticles of diameter between about 60 nm and about 400 nm.
 2. The method of claim 1, wherein the active ingredient is hydrophobic.
 3. The method of claim 1, wherein the subunit nanoparticles are made by a method comprising: dissolving the drug in aqueous solution; adding the aqueous solution to an agitated organic polymer solution to form a water-in-oil emulsion; adding the solution to an excess of water including an emulsifier with agitation to form water-in-oil-in-water emulsion; removing the organic solvent.
 4. The method of claim 1, wherein the subunit nanoparticles are made by a method comprising: dissolving the drug in volatile water-miscible solvent to form a solution; adding the solution to an agitated aqueous solution of carrier polymer to form an oil-in-water emulsion; and diluting the oil-in-water emulsion with water; and filtering the solution to collect the nanoparticles.
 5. The method of claim 1, wherein the subunit nanoparticles are made by a method comprising: dissolving the drug in a water immiscible volatile organic solvent which includes a carrier polymer to form a solution; adding the solution to an excess of water which includes an emulsifier; and removing the solvent.
 6. The method of claim 1, wherein the subunit nanoparticles are made by a method comprising: grafting the drug to a water soluble polymer; and aggregating the modified polymer.
 7. The method of claim 1, wherein the composite nanoparticles are made by a method comprising: dispersing the subunit nanoparticles in water; adding the dispersed nanoparticles to an agitated water immiscible organic solvent which includes a carrier polymer to form a water in oil solution; adding the water in oil solution to an excess of water which includes an emulsifier; and removing the solvent.
 8. The method of claim 1, wherein the composite nanoparticles are made by a method comprising: dissolving the subunit nanoparticles in a water immiscible volatile organic solvent which includes a carrier polymer to form a solution; adding the solution to an excess of water which includes an emulsifier; and removing the solvent.
 9. The method of claim 1, wherein the composite nanoparticles are crosslinked.
 10. The method of claim 1, wherein the composite nanoparticles are made by a method comprising crosslinking subunit nanoparticles.
 11. The method of claim 1, wherein the composite nanoparticle decomposes in tumor tissue.
 12. The method of claim 1, wherein the composite nanoparticle decomposes below about pH 6.4.
 13. The method of claim 1, wherein the composite nanoparticle disintegrates into subunit particles at the pH of the interstitial space of the tumor tissue, wherein the pH of the cancer interstitial space is between 5.6 and 6.4. 